The objective of this project is to apply the techniques of the in vitro motility assay to investigate regulation of cardiac muscle contraction. Using the in vitro motility assay, one can test in a simplified system with well defined proteins whether Ca2+ regulates crossbridge (XBr) kinetics as suggested by some skinned and intact muscle studies and test which specific proteins are responsible for the observed differences in regulation between cardiac and skeletal muscle. It will also be possible to test aspects of regulation which might depend on the specific arrangement of filaments in the sarcomere and test whether XBr's can activate the thin filament, producing superactivation. Although the in vitro motility assay has been used for some time now to investigate the properties of molecular motors, this is the first direct application to the study of regulation in the actin-myosin system under more physiological conditions.